A-chain isozymes of recombinant and natural urokinases: Preparation, characterization, and their biochemical and fibrinolytic properties

Recombinant two-chain urokinase has been prepared by careful activation of r-single chain pro-urokinase with human plasmin, and has been characterized by electrophoretic, composition, and sequence analyses. We determined that urokinase prepared in this manner was >90% high molecular weight and was comprised of predominantly (88%) A-chain C-terminal lysine (amino acid #158), with some C-terminal arginine (derived from amino acid #156). Treatment of the recombinant product (termed LYS-Urokinase) with carboxypeptidase B removed the lysine and resulted in a second urokinase preparation (termed PHE-Urokinase), comprised mostly (85%) of C-terminal phenylalanine (derived from amino acid #157), with lesser amounts of C-terminal arginine and lysine. These isozymes have been characterized by digestion of the isolated A-chains with endoproteinase Glu-C (Staph V8 protease), which results in peptides beginning with Leu(144) and ending with an amino acid characteristic of the C-terminus of the A-chain. The reference peptides were derived from the A-chain of low molecular weight urokinase (Abbokinase®), and were identified as Leu(144) to Arg(156), Leu(144) to Phe(157), and Leu(144) to Lys(158). Several preparations of LMW-urokinase have been found to have these three A-chain peptides in varying amounts which, after reduction and carboxymethylation of the enzyme, can be isolated and quantified by reversed phase high pressure liquid chromatography. The two isozymes of recombinant urokinase (LYS-Urokinase and PHE-Urokinase) have been found to be equipotent in an in vitro clot lysis assay system utilizing human plasma and in an in vivo canine femoral artery clot lysis model. The enzymes also had the same specific activity when assayed with the chromogenic substrate S-2444. We have also characterized four commercial preparations of clinical grade urinary urokinase and the high molecular weight international urokinase standard. All were found to be heterogeneous in the composition of the C-terminus of the A-chain, with Phe(157) as the predominant C-terminal amino acid.